Iterative model building, structure refinement and density modification with the<i>PHENIX AutoBuild</i>wizard

Terwilliger, Thomas C.; Grosse‐Kunstleve, Ralf W.; Afonine, Pavel V.; Moriarty, Nigel W.; Zwart, Peter H.; Hung, Li Wei; Read, Randy J.; Adams, Paul D.

doi:10.1107/s090744490705024x

Cited by 1,411 publications

(1,076 citation statements)

References 28 publications

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“…We built an initial model using RESOLVE 31 and manually completed the model using Coot 32 . As we obtained the native dataset from a merohedrally twinned crystal (apparent spacegroup P6), we refined the structure using the native dataset in spacegroup P3, applying the respective twin-operator in PHENIX [33][34][35] . We determined the structure of pre-cleavage endoNF-CIMCD by molecular replacement using the β-helix domain of endoNF , PDB 1V0E 7 and the structure of GP12-CIMCD as a search model in MOLREP 36 and PHASER 37 .…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of an intramolecular chaperone mediating triple–β-helix folding

Schulz

Dickmanns

Urlaub

et al. 2010

Nat Struct Mol Biol

106

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Proteins fold into their functional three-dimensional structure based on the information encoded in the residue sequence 1 . In all domains of life, various strategies evolved to assist folding processes in order to prevent immediate misfolding and aggregation of the nascent polypeptide chain. In the crowded cellular environment, protein folding is often aided by molecular chaperones. Molecular chaperones differ in size, function and energy dependence; however, all have in common to bind to the unfolded state of the protein in order to facilitate or mediate assembly of the correct three-dimensional structure 2,3 . In contrast to molecular chaperones, the intramolecular chaperones (IMCs) constitute a different class of chaperones. As part of the polypeptide chain, the IMC is typically cleaved off the target protein after the folding process is completed. Two classes of IMCs can be distinguished: class I IMCs assist the protein to fold into the correct tertiary structure, whereas class II IMCs are involved in quaternary structure assembly 4 . In contrast to many molecular chaperones, no evidence for an ATP-driven cleavage reaction could be found in IMCs. An example of a class II intramolecular chaperone has been identified in viral tailspike and fiber proteins. These proteins are functionally unrelated but share a highly conserved chaperone domain at their C terminus (C-terminal intramolecular chaperone domain, CIMCD), which is cleaved at a conserved position in an autoproteolytic reaction 5 . It was shown that the covalent linkage between the CIMCD and N-terminal pre-protein is necessary for correct folding, indicating that an in trans function of the chaperone is impossible 5 . Furthermore, it could be shown that some of the chaperone domains are exchangeable between the different preproteins 5,6 . While the three-dimensional structures of the CIMCDs are as yet unknown, the crystal structure of N-terminally truncated mature endoNF shows that the homotrimeric enzyme comprises a triple β-helix involved in substrate recognition 7 . This triple β-helix and the related triple-β-spiral motif have been identified in various proteins, which often play a role as virulence factors 8 . In triple β-helices, three polypeptide chains wind around a common threefold symmetry axis, conferring an extraordinary stability to the protein. The rigid elongated shape allows triple β-helix-comprising proteins to protrude from a pathogen's surface in order to interact with flexible host cell receptors, like lipopolysaccharides 9 . However, proper assembly of triple-β-helical folds poses to be difficult in the absence of a trimerization domain 10 . Hence, most triple β-helices depend on a C-terminal extension for trimerization and correct assembly 11 .Here we present the crystal structures of two representatives of a large group of systematically, functionally and structurally similar intramolecular chaperones: the Escherichia coli phage K1F endosilidase CIMCD and the Bacillus subtilis phage GA-1 neck appendage protein CIMCD. Furthermore...

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Section: Methodsmentioning

confidence: 99%

Crystal structure of an intramolecular chaperone mediating triple–β-helix folding

Schulz

Dickmanns

Urlaub

et al. 2010

Nat Struct Mol Biol

106

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“…Data were collected on crystals flash frozen in liquid nitrogen via the oscillation method and were reduced using the HKL2000 program suite (72). Phases were calculated via molecular replacement (MR) using Phaser as implemented in AutoMR, a component of the PHENIX software package (45,(73)(74)(75). For our initial structure of apo-hFRα (P6 5 form), model coordinates for chicken riboflavin binding protein (chRfBP) were kindly provided by H. Monaco (University of Verona, Verona, Italy), and a truncated form of the cRfBP model was used as a MR search model in Phaser (57).…”

Section: Methodsmentioning

confidence: 99%

Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition

Wibowo

Singh

Reeder

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

174

190

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Antifolates, folate analogs that inhibit vitamin B 9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.isothermal titration calorimetry | targeted drug delivery

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“…Thirteen iodine sites were found using AUTOSOL (49), as implemented from PHENIX (50), with a figure of merit (FOM) of 0.39. A model, which was >90% complete, was built using AUTOBUILD (51) and used as a search model for molecular replacement of the native data (Table 1), implemented with PHASER (52), from within PHENIX (50). Clear and unambiguous solutions were found to the rotation and translation functions, and the resulting model used for automated building by AUTOBUILD, followed by several cycles of refinement, using phenix.refine (53) combined with manual building using the molecular graphics package COOT (54).…”

Section: Methodsmentioning

confidence: 99%

Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis

Karuppiah

Collins

Thistlethwaite

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Significance Type IV pili are long, thin fibers, formed mainly of polymers of a single pilin protein, which are displayed on the surfaces of many bacteria, including several human pathogens. Here, we report three-dimensional reconstructions of the PilMNO inner membrane complex, alone and in complex with pilin protein, through a combination of X-ray crystallography and electron microscopy. PilMNO forms a dimeric T-shaped structure, binding two copies of the pilin protein at its extremities. The results provide a structural model for the way in which pilin is harvested from the inner membrane and made available to other components of the type IV pilus biogenesis machinery.

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Iterative model building, structure refinement and density modification with thePHENIX AutoBuildwizard

Cited by 1,411 publications

References 28 publications

Crystal structure of an intramolecular chaperone mediating triple–β-helix folding

Crystal structure of an intramolecular chaperone mediating triple–β-helix folding

Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition

Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis

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